NQO1 (NAD(P)H:quinone oxidoreductase 1, DT-diaphorase) has attracted considerable attention because of its ability to bioactivate antitumor quinones. The elevated NQO1 content throughout many solid tumors has led to an interest in designing antitumor quinones which can be bioactivated by NQO1 for therapeutic purposes. In this application. we propose to focus on the role of NQO1 in-the bioactivation of antitumor quinones and on j the mechanisms underlying the null NQO1 activity in individuals carrying a polymorphism in NQO1. The implications of the null polymorphism for therapy using NQO1-directed antitumor agents will also be defined. We will determine the ability of novel antitumor quinones to serve as substrates for human NQO1 using both biochemical and molecular modeling studies. We will also test the hypothesis that compounds efficiently bioactivated by NQO1 will induce selective DNA damage and cytotoxicity in a panel of cell lines stably transfected with hNQO1 and examine which solid tumors contain elevated levels of NQO1 by immunohistochemistry. It is also critical to understand the mechanisms underlying the elevated NQO1 expression in tumors and we will identify cis acting DNA sequences and traps acting nuclear protein-DNA interactions that modulate expression of NQO1 . We will define the role of the ubiquitin/proteasome system in degradation of mutant NQO1 protein and test the hypothesis that the mutant NQO1 protein is misfolded as a result of the homozygous mutation. The null polymorphism in NQO1 also gives us a unique opportunity to test the hypothesis that clinical response to antitumor quinones depends on NQO1 genotype and phenotype. This hypothesis will be examined using primary cultures of human tumors, archived tissue from previous clinical studies and by measuring molecular correlates of response and/or toxicity in a Phase I trial of a novel antitumor quinone developed in our laboratory during the last grant period. The proposed studies represent an integrated chemical, biochemical and molecular approach to determine the significance of NQO1 and NQO1-directed antitumor agents in chemotherapy.